Methods for producing and transforming cassave protoplasts

ABSTRACT

The invention relates to a method for producing protoplasts of cassava or closely related species, which protoplasts are capable of regeneration into plants. The method comprises producing friable embryogenic callus from explants of cassava or closely related species and isolating protoplasts from said friable embryogenic callus. The invention also concerns protoplasts obtainable by said method. The invention further relates to a method for transforming such a protoplast of cassava or closely related species, and transformed protoplasts obtainable thereby. In addition, the invention concerns a method for regenerating plants from these protoplasts and a cassava plant or closely related species obtainable thereby.

This application claims the benefit of International Application NumberPCT/NL97/00285 filed on May 20, 1997 which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

Genetic modification or transformation is the technique wherein one or afew gene(s) are added to a commercial interesting genotype or clone. Inprinciple a successful transformation system requires an efficientsystem where new plants are formed from specific plant parts (stem,leaf, node and so on) or from protoplasts (single cells without cellwall) derived of these parts, a system to transfer DNA molecules to theplant's parts or protoplasts and a system to select tissue and plantswhich contain and express the introduced gene(s).

In principle protoplasts are the most ideal system for DNA delivery.They can be cultured as single cells that produce multicellular coloniesfrom which plants develop. Plants derived from protoplasts are generallyclonal in origin. This provides a useful tool for any transformationsystem, because it will eliminate chimerism in transgenic plants.

Cassava is very recalcitrant for plant regeneration of protoplasts.There is only one report of shoot regeneration from protoplasts ofcassava (Shahin and Shephard, 1980). They used well expanded leaves forthe isolation of protoplasts. Despite considerable efforts, plantregeneration from protoplasts (isolated from leaves, stems, and roots)has never been repeated since then (Anonymus, 1985; Nzoghe, 1991;Anthony et al., 1995, Sofiari, 1996). A logical approach was to usetissues which contain embryogenic cells. Such cells are found in theapical meristems, young leaves or somatic embryos cultured on auxinsupplemented media (Stamp and Henshaw, 1987a; Raemakers et al., 1993a).However, protoplasts isolated from these tissues gave in the best casegreen callus and adventitious roots (Sofiari, 1996). Recently, a newtype of somatic embryogenesis was developed. In this in vitro system theembryos do not develop beyond the (pre-)globular stage and theembryogenic callus is highly friable (Taylor et al., 1995). Transfer ofthis friable embryogenic callus (FEC) to liquid medium resulted in asuspension-like culture. In leek (Buitenveld and Creemers, 1994),petunia (Power et al., 1979), rice (Kyozuka et al., 1988), sugarcane(Chen, et al., 1988), and wheat (Chang et al., 1991) such cultures werean excellent source for protoplast regeneration.

We have now found that in cassava FEC is the only tissue from whichprotoplasts can be isolated which are able to regenerate into plantssofar.

SUMMARY OF THE INVENTION

Thus the present invention provides a method for producing protoplastsof cassava or a closely related species, which protoplasts are capableof regeneration into plants, comprising producing friable embryogeniccallus from explants of cassava or a closely related species andisolating protoplasts from said friable embryogenic callus. It appears,as will be described below, that for obtaining suitable protoplasts theculture in solution of the FEC is quite important. Therefore the presentinvention further provides a method wherein the friable embryogeniccallus is subjected to culture in a liquid medium.

Protoplasts are preferably produced by subjecting plant cells toenzymatic breakdown of the cell walls. The invention thus provides amethod whereby a mixture of cell wall degrading enzymes, such as acellulase, a pectolyase and/or a macerozyme are used to produceprotoplasts.

It also appears that the method according to the invention works bestwhen the plants from which explants are to be taken are pretreated.Therefore the invention provides a method whereby the plants from whichexplants are taken are pretreated with an auxin as described below.

On the explants preferably embryogenesis is induced resulting in aninvented method whereby the friable embryogenic callus is produced fromtorpedo shaped primary or mature embryos. The reason is explained in thedetailed description. Protoplasts obtainable by a method as disclosedabove are also part of the invention.

An important reason for wanting to have protoplasts which can beregenerated into plants is of course that protoplasts can be easilytransformed or transduced or provided with additional geneticinformation by any other suitable method. Thus one is now able toprovide cassava plants or closely related species with genetic materialof interest. The invention thus also provides a method for transforming(defined as providing with in any suitable manner) a protoplast of acassava or a closely related species by providing said protoplast withadditional genetic information through infection by a bacteriumcomprising said additional genetic information such as Agrobacteriumtumefaciens, by electroporation or chemical poration providing a vectorcomprising said additional genetic information or by particlebombardment whereby the particles are coated with the additional geneticinformation, whereby a protoplast obtainable from friable embryogeniccallus is transformed. The invention also encompasses transformedprotoplasts obtainable by such a method.

Below a short introduction is given on the usefulness of transformingplants, such as cassava.

Application of plant gene technology encompasses a multitude ofdifferent techniques ranging from isolation of useful genes, theircharacterization and manipulation, to the reintroduction of modifiedconstructs into the plants (Lonsdale, 1987). Plant gene technology willcatalyze progress in plant breeding, as is exemplified by a few examplesof transgenic crops like rice (Chen et al., 1987; Shimamoto et al.,1989), maize (Gordon-Kamm et al., 1990; Vain et al., 1993), wheat (Markset al., 1989), and potato (De Block, 1988; Visser et al., 1989). Rapidprogress in gene technology has allowed insight into the complexmolecular mechanism of plant pathogen recognition and the naturaldefence strategies of host plants. This technology can also be used forcontrolled and efficient identification of desirable genotypes, farbeyond the possibilities of classical breeding.

For instance electroporation of protoplasts derived from suspensioncultures led to the transformation of maize (Rhodes et al., 1988), rice(Toriyama et al., 1988) and orchardgrass (Horn et al., 1988).

Successful attempts have been made to improve resistance againstpathogenic viruses like tobamovirus in tobacco (Powel Abel et al.,1986), potexvirus in potato (Hoekema et al., 1989) and in papaya (Fitchet al., 1992). In the above examples the introduced trait was based onexpressing of single genes that are coding for the coat protein. Incassava, African cassava mosaic virus (ACMV) and cassava common mosaicvirus (CCMV) may be controlled by the coat protein-mediated resistancetechnique (Fauquet et al., 1992). The genes encoding key enzymes ofcyanogenesis have been cloned (Hughes et al., 1994) which makesmanipulation of cassava cyanogenesis by genetic transformation using theantisense approach feasible. Another embodiment of the invention is themanipulation of starch in the cassava tubers.

Thus the present invention provides a transformed protoplast whereby theadditional genetic information comprises an antisense construct,particularly one whereby the antisense construct is capable ofinhibiting the amylose synthesis pathway.

A protoplast cannot grow in the field, nor can it be harvested. Thoughprotoplasts are necessary for transformation, it must be possible toregenerate said protoplasts into embryos and/or plants. This is a veryimportant embodiment of the invention, because cassava has been shown tobe difficult to regenerate from protoplasts. The detailed descriptionexplains how this may be achieved. For further information reference ismade to the thesis written by E. Sofiari titled Regeneration andTransformation of Cassava (Manihot Esculenta Crantz.), a copy of whichis enclosed with the present application, which is as yet unpublishedand which is incorporated herein by reference. Thus the inventionprovides a method for regenerating plants from protoplasts whereby aprotoplast according to the invention is induced to produce an embryo,which embryo is consequently induced to produce a plant.

The plants obtainable by said method are also part of the invention, inparticular plants whereby the tubers contain essentially no amylose.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Schematic representation of somatic embryogenesis in cassava,including primary, secondary somatic embryogenesis, selection of friableembryogenic callus, maturation and desiccation followed by germination.

gd2=medium supplemented with Gresshoff and Doy salts (1974) and vitaminsplus 20 g/l sucrose.

gd4=medium supplemented with Gresshoff and Doy salts (1974) and vitaminsplus 40 g/l sucrose.

ms2=medium supplemented with Murashige and Skoog salts and vitamins plus20 g/l sucrose.

pic=10 mgl/l Picloram, NAA=10 mg/l naphthalene acetic acid, 2,4-D=8mg/l, 2,4-dichiorophenoxy acetic acid.

sh6=medium supplemented with Schenk and Hildebrandt (1972) salts andvitamins plus 60 g/l sucrose.

Detailed Description

Initiation of FEC

The procedure to obtain FEC is outlined in FIG. 1. It starts with theinduction of primary embryos. Primary embryos are formed in a two stepprocedure. In the first step explants are cultured on mediumsupplemented with salts and vitamins (preferably Murashige and Skoog(1962)), a carbohydrate source (for example 20 g/l sucrose) and an auxin(e.g. 1-8 mg/l picloram, or dicamba or 2,4-D) for the initiation ofembryos. After 10 to 15 days on this first medium bipolar torpedo shapedembryos are formed. Torpedo shaped embryos possess a clear hypocotyl andcotyledon primordia. After transfer of the explants with torpedo shapedembryos to a step 2 medium (the same medium as step 1 but without anauxin) the torpedo shaped embryos become mature. Mature embryos possesslarge green cotyledons.

Zygotic embryos (Stamp and Henshaw 1982; Konan et al., 1994), young leafexplants or apical meristems (Stamp and Henshaw, 1987a; Szabados et al.,1987; Mroginsky and Scocchi, 1993; Raemakers 1993a; Narayanaswamy etal., 1995) and floral tissue (Mukherjee, 1995) can be used to obtainprimary embryos. In this way many different genotypes were evaluated fortheir ability to form primary embryos. In this protocol primary somaticembryos were only formed after culture on solid medium and never afterculture in liquid medium. Furthermore, somatic embryos (primary) wereonly observed if the auxins Picloram, Dicamba or 2,4-D were used and notwith IAA, IBA or NAA.

In the presently used protocol there is genotypic variation in thenumber of mature embryos formed per cultured explant. The genotypesM.Col1505, M.Col22 and Gading gave the highest numbers of mature embryosper cultured leaf explant (ME/CLE). However, the number of matureembryos formed was low. In M.Col22 a maximum of 22% of the leaf explantsisolated from in vitro grown plants and cultured on a step 1 medium with4 mg/l 2,4-D, formed ME with a maximum number of 0.8 ME/CLE. On a step 1medium with 8 mg/l 2,4-D a maximum of 49% of the leaf explants formed MEwith a maximum number of 3.5 ME/CLE. Higher 2,4-D concentrations did notfurther improve the embryogenic capacity of explants.

In an attempt to improve the capacity of leaf explants to produceprimary somatic embryos, donor plants were grown under differentconditions. Growth of in vitro donor plants under different lightregimes (8, 12, 16 or 24 hours) had no influence on the embryogenicresponse. However, a reduction of the light intensity had a positiveeffect. The best results were obtained with leaf explants isolated ofdonor plants grown at 8 μEm⁻²s⁻¹ and cultured on a step 1 medium.

Other investigators have shown that in certain genotypes, Dicamba (1-66mg/l) and Picloram (1-12 mg/l) are superior to 2,4D for inducing primaryembryogenesis (Ng 1992; Sudarmonowati and Henshaw, 1993; Taylor andHenshaw, 1993). Mathews et al. (1993) improved the efficiency of primaryembryogenesis in the genotype M.Col1505 by transferring explants after15 days of step 1 medium to a growth regulator-free medium supplementedwith 0.5% charcoal. On this medium maturation was improved and as aresult the number of mature embryos increased from 0.4 in the control to3.4 ME/CLE.

The best results were obtained if donor plants were pretreated withauxins as 2,4-D or picloram or Dicamba. For this, plants were grown inliquid MS20 medium and supplied with the auxin (final concentration 8mg/l) after 12 days of growth. Two days later leaf explants wereisolated of the donor plants and cultured on step 1 medium with 8 mg/l2,4-D, picloram or Dicamba. In the clone M.Col22 this resulted in aproduction of 9.4 ME/CLE. This was significantly higher than in theH₂O-treated control-plants, where 3.5 ME/CLE were produced (Table 3).

The general applicability of the auxin pretreatment was tested onseveral different genotypes. Without a pretreatment of donor plants twogenotypes formed ME and at low frequency. After a pretreatment of donorplants, leaf explants of almost all genotypes formed ME.

Eventually we were able to obtain mature primary somatic embryos from 18of the 22 tested genotypes (Table 1, except TMS30221, TMS30001, TMS30572and Sao Paolo).

Primary somatic embryos derived from zygotic embryos and from leaveshave been used as explants to initiate secondary embryos (Stamp andHenshaw, 1987b; Szabados et al., 1987; Mathews et al., 1993; Raemakerset al., 1993bc; Luong et al., 1995). Continuous culture of somaticembryos on auxin supplemented medium resulted in a cyclic system ofsomatic embryogenesis. The way of subculturing somatic embryos forsecondary embryogenesis seemed to influence the morphology of theembryogenic tissue. Clumps of somatic embryos recultured monthly onsolid 2,4D containing medium in the dark developed into finger-likeembryo initials formed on the top of older embryos. The embryos did notpass the torpedo-shaped stage.

Further development occurred if the clumps with embryos were transferredto step 2 medium in the light (Szabados 1987). Normally mature somaticembryos were cultured in step 1 medium in the light and twenty dayslater the explants were transferred to step 2 medium for maturation. Inthis system embryos developed to maturity and mature embryos with largegreen cotyledons were used to start a new cycle of embryogenesis whereasin the system of others torpedo shaped embryos were used to start a newcycle of secondary somatic embryogenesis.

This system of multiplication of mature embryos has been tested in 14 ofthe in Table 1 mentioned genotypes. Despite the fact that in mostgenotypes only a few mature primary embryos were available, allgenotypes, except one, gave new mature embryos after culture on 2,4-Dsupplemented medium, in a much higher frequency as observed for primarysomatic embryogenesis. Embryogenicity was maintained by regularsubculture of mature embryos for more than one year (Szabados et al.,1987; Mathews et al., 1993; Raemakers, 1993). New somatic embryos wereformed both in liquid and solid medium. In all the genotypes it wasobserved that in liquid medium more embryos were formed than in solidmedium and that fragmentation of embryos before the start of a new cycleof secondary somatic embryogenesis increased the production compared towhole embryos. In for example M.Col22 whole embryos cultured on solidmedium produced 8 embryos per cultured embryo, whereas fragmentedembryos cultured in liquid medium produced 32 embryos per culturedembryo (Raemakers et al., 1993c). Not only, 2,4-D, Picloram and Dicamba,but also NAA had the capacity to induce secondary embryogenesis. IBA andIAA did not induce secondary embryogenesis. NAA has been usedsuccessfully in Adira 1, Adira 4, Gading, Line 11, M.Col22, M.Col1505,TMS90853 and Gading. In general more mature embryos were produced in NAAsupplemented medium than in 2,4-D, Picloram or Dicamba supplementedmedium. Furthermore, the development of NAA induced embryos was fasterthan with 2,4-D, Dicamba or Picloram. Shortening the culture durationhas a beneficial effect, particularly, when operating on a large scale.

Histologically, the by 2,4-D newly induced secondary embryos wereattached vertically to the explants whereas those by NAA werehorizontally.

There is still a problem in obtaining embryogenic cultures of cassava(Mroginski and Scocchi, 1992; Taylor et al., 1992; Narayanaswamy et al.,1995; Sudarmonowati and Bachtiar, 1995). The main problem is not thatembryogenic tissue from primary explants can be obtained, but the largescale multiplication of this tissue by secondary embryogenesis. For thispurpose, either tissue consisting of torpedo shaped embryos or matureembryos can be used. Multiplication of torpedo shaped embryos is highlygenotype dependent, while multiplication of mature embryos is largelygenotype independent (Raemakers, 1993). Both primary and secondarysomatic embryogenesis are characterized by the formation of propaguleswith a bipolar structure. These bipolar torpedo shaped embryos arealready formed on the auxin supplemented step 1 medium. Therefore,Taylor et al., (1995) proposed the term organized embryogenesis.Organized cells are defined as a group of actively dividing cells,having the tissues and organs formed into a characteristic unified whole(Walker, 1989).

A less organized type of somatic embryogenesis was developed by Tayloret al. (1995). With continuous selection, organized embryogenic tissuecultured on a Gresshoff and Doy (1972) medium salts and vitaminssupplemented with 10 mg/l Picloram (GD2) converted gradually into a lessorganized tissue. This tissue consisted of a callus-like mass of(pro-)globular embryos which was very friable. Therefore, this tissuewas called friable embryogenic callus (FEC). The cells in FEC arecontinuously in a state where they break away from group control andbecause of that they are not organized into a unified structure. FEC ismaintained on a medium consisting of Gresshoff and Doy (1972) vitaminsand salts, 7 g/l Daichin agar, 20 g/l sucrose and 10 mg/l Picloram(solid GD2). Every three weeks the friable embryos were subcultured onthe above mentioned medium. In order to initiate liquid suspensioncultures 0.5 g of friable embryos was transferred in a flask of 200 mlwith 50 ml of liquid medium supplemented with Schenk and Hildebrandt(1972) salts and vitamins, 60 g/l sucrose and 10 mg/l Picloram (liquidSH6). The medium was refreshed every 2 days and after 14 days thecontent of each flask was divided over 5 new flasks. The pH was adjustedto 5.7 before autoclaving. The temperature in the growth chamber was 30°C., the photoperiod 12 hours and the irradiance 40 μmolm⁻²s⁻¹.Suspension cultures were initiated by culturing FEC in Schenk andHildebrandt (1972) medium supplemented with 6% (w/v) sucrose and 10 mg/lPicloram (SH6). Every 2-3 days this medium was refreshed.

To keep a culture in a highly friable state the FEC has to be sievedonce in two months. In practice the part of the FEC which will gothrough a sieve with a mesh of 1 mm² will be used for subculture.

FEC will almost never form torpedo shaped embryos on the GD2 or in SH6medium. Torpedo shaped and subsequent mature embryos are formed if FECis cultured on maturation medium. Maturation medium consist of Murashigeand Skoog (1962) salts and vitamins, 0.1 g/l myo-inositol, 20 g/lsucrose, 18.2 g/l mannitol, 0.48 g/l MES, 0.1 g/l caseinhydrolysate,0.08 g/l adenosine sulphate, 0.5 mg/l d-calcium-panthotenate, 0.1 mg/lcholine chloride, 0.5 mg/l ascorbic acid, 2 mg nicotinic acid, 1 mg/lpyridoxine-HCl, 10 mg/l thiamine HCl, 0.5 mg/l folic acid, 0.05 mg/lbiotin, 0.5 mg/l glycine, 0.1 mg/l L-cysteine, 0.25 mg/l riboflavine and1 mg/l picloram. This maturation medium was refreshed every 3 weeks.

Mature embryos could be induced into secondary somatic embryogenesis byculturing on MS20 medium supplemented with 2,4-D, picloram, Dicamba orNAA. Primary and secondary somatic embryogenesis are relatively easy toestablish in a wide range of genotypes (see Table 1), while FEC is forthe time being restricted to a few genotypes. The prospect of FEC for anew system of somatic embryogenesis and genetic transformation ispromising, although further research is needed to make this systemapplicable to more genotypes. Essential for this process is theavailability of high quality organized tissue and the ability of thistissue to convert into FEC. Taylor et al. (1995) “used organizedembryogenic tissues” which were multiplied at the torpedo shaped stateto initiate FEC. In this case two steps (initiation of organized. tissueand conversion into unorganized tissue) are determinative for thesuccessful initiation of FEC. Both steps are genotype dependent. Iforganized tissue is multiplied in the mature state as described byRaemakers (1993) then only the ability of this tissue to convert intoFEC is a determinative step to initiate FEC. It remains to beinvestigated whether or not organized tissue can be used as startingmaterial. If organized tissue cannot be used, then this tissue should befirst multiplied in the immature state before it can be used to initiateFEC. This is readily accomplished by, either culturing explants at ahigh density or by reducing the cyclic duration.

Regeneration of Plants from Protoplasts

Isolation of Protoplasts

For protoplast isolation both FEC cultured on solid GD2 or liquid SH6can be used. However, the highest yield of protoplasts was obtained fromFEC which has been cultured for 1 to 3 weeks in liquid SH6.

Two gram of FEC was placed in Petri dishes (Ø 9 cm) containing 10 ml ofcell wall digestion solution. Cell wall digestion solution consisted ofa mixture of cell wall degrading enzymes; 10 mg/l pectolyase, 10 g/lcellulose, 200 mg/l macero enzym growth regulators (NAA 1 mg/l, 2,4-D 1mg/l, Zeatin 1 mg/l); major salts (368 mg/l CaCl₂; 34 mg/l KH₂PO₄; 740mg/KNO₃; 492 mg/l MgSO₄.7H₂O); minor salts (19.2 mg/l NA-EDTA; 14 mg/lFeSO₄.7H₂O) and osmoticum (91 g/l D-mannitol) and 0.5 g/l MES. The cellwall degrading enzymes cellulase (1-10 g/l) plus Macerozyme (200 mg/l)were successful for protoplast isolation. The extra addition ofPectolyase (0.001-0.01 g/l) and/or Driselase (0.02 g/l) increased theyield of protoplasts. After 18 h of incubation, 10 ml of washing mediumwas added to the solution. Washing medium with an osmolarity 0.530mOsm/kg consisted of major salts (see cell wall digestion solution),45.5 g/l mannitol and 7.3 g/l NaCl. The digested tissue was filteredthrough a 73 μM pore size filter (PA 55/34 Nybolt—Switzerland) into a250 ml beaker glass. The filtrate was divided equally over two 12 mlconical screw cap tubes, and centrifuged at 600 rpm for 3 min. (Mistral2000). The washing procedure was repeated once after removal of thesupernatant. The protoplast solution was resuspended by floating on 9.5ml solution containing major and minor salts (see cell wall digestionsolution) and 105 g/l sucrose. The pH was 5.8 and the osmolarity 0.650mOsm. The solution with protoplasts was allowed to equilibrate for 5minutes before 0.5 ml of washing medium was gently added on the top.After centrifugation at 700 rpm for 15 min. (Mistral 2000), theprotoplasts were concentrated in a band between the sucrose and washingmedium. The protoplast layer was harvested with a pasteur pipette andthe yield was counted in a standard haemocytometer chamber.

Protoplast Culture

Protoplasts were cultured in media solidified with agarose 0.2% w/v(Dons en Bouwer, 1986) in petri dishes containing 10 ml of the sameliquid medium. The following media resulted in the formation of microcallus:

TM2G medium (Wolters et al., 1991) supplemented with only auxins (0.1-10mg/l NAA or 0.1-10 mg/l Picloram, or 0.1-10 mg/l IAA, or 0.1-10 mg/l2,4-D, or 0.1-10 mg/l Dicamba, or 0.1-10 mg/l, or 0.1-10 mg/l) or auxinsplus cytokinins (0.01-1 mg/l zeatin, 0.01-1 mg/l 2-iP, 0.01-1 mg/l BA,0.01-1 mg/l TDZ, 0.01-1 mg/l kinetin).

medium A (Murashige and Skoog (1962) salts and vitamins, 4.5 g/lmyo-inositol, 4.55 g/l mannitol, 3.8 g/l xylitol, 4.55 g/l sorbitol,0.098 g/l MES, 40 mg/l adeninsulphate and 150 mg/l caseinhydrolysate,0.5 mg/l d-calcium-panthotenate, 0.1 mg/l choline-chloride, 0.5 mg/lascorbic acid, 2.5 mg/l nicotinic acid, 1 mg/l pyridoxine-HCl, 10 mg/lthiamine-HCl, 0.5 mg/l folic acid, 0.05 mg/l biotine, 0.5 mg/l glycine,0.1 mg/l L-cysteine and 0.25 mg/l riboflavine and 59.40 g/l glucose)supplemented with only auxins (0.1-10 mg/l NAA or 0.1-10 mg/l Picloram,or 0.1-10 mg/l IAA, or 0.1-10 mg/l 2,4-D, or 0.1-10 mg/l Dicamba pluscytokinins (0.01-1 mg/l zeatin, 0.01-1 mg/l 2-iP, 0.01-1 mg/l BA, 0.01-1mg/l TDZ, 0.01-1 mg/l kinetin).

The media were refreshed every 10 days, by replacing 9 ml with freshmedium. After two months of culture in the first medium, high qualityFEC was selected and either culture for further proliferation or formaturation. For proliferation FEC was transferred to Gresshoff and Doy(1974) medium supplemented with 40 g/l sucrose, 7 g/l Daichin agar and 2mg/l picloram (GD4). After 3 weeks the FEC was transferred to aGresshoff and Doy medium supplemented with 20 g/l sucrose, 7 g/l agarand 10 mg/l Picloram (GD2). Suspension cultures ware initiated bytransferring 1.0 g of FEC to liquid SH6% medium supplemented with 10mg/l Picloram. Two weeks later the suspension was divided over newflasks with an initial packed cell volume of 1.0 ml.

After 2 months of culture, 10⁴ protoplasts cultured in TM2G supplementedwith 0.5 mg/l NAA and 1 mg/l Zeatin at a density of 10⁵/ml produced 1058micro-calli, whereas 10⁴ protoplasts cultured at a density of 10⁶/mlonly produced 64 micro-calli.

Replacing TM2G medium with medium A reduced at both densities the numberof micro-calli significantly. At this stage at least three types ofcalli could be distinguished. One type consisted of globular shapedembryos which were mostly observed in protoplasts cultured at a densityof 10⁶. Some of them developed cotyledon like structures, light green incolor. However, these embryos could not be germinated properly. Anothertype was fast growing and consisted of a large compact callus, they wereobserved in protoplast cultures of both densities. This callus neverdeveloped embryos. The third type was highly friable callus and wasobserved at both densities. At a density of 2-5×10⁵ (medium TM2G) about60% of the calli were friable and embryogenic. The FEC was eithersubcultured for further proliferation or for maturation.

Proliferation of FEC Derived from Protoplasts

Following selection of FEC, 0.1 g of it cultured for three weeks on GD 4plus 2 mg/l Picloram increased into 0.7 g of tissue. More than 95% ofthe tissue consisted of high quality FEC. Subsequently, this tissue wasmaintained by subcultures of three weeks on GD2 medium supplemented with10 mg/l Picloram. To initiate suspension cultures FEC was transferred toliquid medium. The increase in packed cell volume (PCV) of this materialwas slightly higher than that of the original material (data not shown).

Maturation of FEC Derived from Protoplasts

In an attempt to induce maturation of embryos, FEC isolated after twomonths of culture in TM2G was cultured on maturation medium. Maturationmedium consisted of Murashige and Skoog (1962) salts and vitamins, 0.1g/l myo-inositol, 20 g/l sucrose, 18.2 g/l mannitol, 0.48 g/l MES, 0.1g/l caseinhydrolysate, 0.08 g/l adenine sulphate, 0.5 mg/ld-calcium-panthotenate, 0.1 mg/l choline chloride, 0.5 mg/l ascorbicacid, 2. mg/l nicotinic acid, 1 mg/l pyridoxine-HCl, 10 mg/l thiamineHCl, 0.5 mg/l folic acid, 0.05 mg/l biotin, 0.5 mg/l glycine, 0.1 mg/lL-cysteine, 0.25 mg/l riboflavine and 1 mg/l picloram. This maturationmedium was refreshed every 3 weeks.

On this medium there is a gradual shift from proliferation tomaturation. As a result the packed cell volume had increased with afactor 4 after two weeks of culture in liquid maturation medium. Alsoafter transfer to solid maturation medium there is proliferation. Aftertwo weeks on solid medium most of the embryos had reached a globularshape and only a few of these globular embryos developed further. Thefirst torpedo shaped embryos became visible after one month of cultureon solid maturation medium. The number of mature and torpedo shapedembryos was not correlated with the plating efficiency but with thedensity of the initially cultured protoplasts. No such embryos wereobtained if protoplasts were cultured on TM2G without growth regulators.The highest number of mature and torpedo shaped embryos was formed fromprotoplasts cultured on TM2G supplemented with 0.5 mg/l NAA and 1 mg/lZeatin. If NAA was replaced by Picloram then the number of torpedoshaped and mature embryos was significantly lower (Table 2). From thetested Picloram concentrations 2 mg/l gave the best results. After 3months of culture between 60 and 200 torpedo shaped and mature embryoswere isolated per agarose drop. Torpedo shaped embryos became mature athigh frequency if they were cultured on fresh maturation medium or onMS2 plus 0.1 mg/l BAP.

Secondary Somatic Embryogenesis and Germination of Mature EmbryosDerived from Protoplasts

Only a few torpedo shaped embryos formed secondary embryos if culturedon liquid or solid MS2 medium supplemented with 10 mg/l NAA or 8 mg/l2,4-D (data not shown). Mature embryos were better explants forsecondary embryogenesis. In both liquid and solid medium 2,4-D wassuperior for induction of secondary embryogenesis as compared to NAA. Ifmature embryos were first cultured in 2,4-D and then in liquid NAA theresponse was comparable with culture in 2,4-D alone. Also embryos whichfirst had undergone a cycle of secondary somatic embryogenesis in mediumwith 2,4-D, produced highly efficient secondary embryos in MS20supplemented with 10 mg/l NAA.

The germination of cyclic or secondary somatic embryos, induced inliquid medium by the auxins 2,4-dichlorophenoxyacetic acid (2,4-D) ornaphthalene acetic acid (NAA), was compared. In all genotypesdesiccation stimulated normal germination of NAA induced embryos.However, the desiccated embryos, required a medium supplemented withcytokinins such as benzytaminopurine (BAP) for high frequencygermination. The morphology of the resulting seedling was dependent onthe concentration of BAP. With 1 mg/l BAP plants with thick and shorttaproots and branched shoots with short intemodes were formed. With 0.1mg/l BAP the taproots were thin and slender and the shoot had only oneor two apical meristems. If the embryos were desiccated sub-optimally,higher concentrations of BAP were needed than if the embryos wereoptimally desiccated to stimulate germination. Also desiccated embryoswhich were cultured in the dark required a lower concentration of BAPand, furthermore, these embryos germinated faster than embryos culturedin the light. Complete plants were obtained four weeks after the startof somatic embryo induction. 2,4-D induced embryos showed a differentresponse. In only one genotype desiccation enhanced germination of 2,4-Dinduced embryos and in three other genotypes it did not. In allgenotypes desiccation stimulated root formation. Embryos cultured in thedark formed predominantly adventitious roots, whereas embryos culturedin the light formed predominantly taproots.

Gene Transfer Systems

Over the past years several transfer techniques of DNA to plantprotoplasts have been developed such as silicon fibers (Kaeppler et al.,1990), microinjection (De Laat and Blaas, 1987) and electrophoresis(Griesbach and Hammond, 1993). The most commonly used andpotentially-applicable ones are Agrobacterium-mediated gene delivery,microprojectile/particle bombardment and protoplast electroporation.

The Agrobacterium tumefaciens DNA delivery system is the most commonlyused technique. It probably relates to the first invention of DNAdelivery in plants by this method. Initially it was limited to Kalanchoeand Solanaceae, particularly tobacco. Nowadays, the use ofAgrobacterium-mediated transformation has changed dramatically, it ispossible to transform a wide range of plants with a limitation inmonocots (reviewed by Wordragen and Dons, 1992).

Although cassava is a host for Agrobacterium it has proven to be nothighly amenable to it.

In principle protoplasts are the most ideal explants for DNA delivery.They can be cultured as single cells that produce multicellular coloniesfrom which plants develop. Plants derived from protoplasts are generallyclonal in origin. This provides a useful tool for any transformationsystem, because it will eliminate chimerism in transgenic plants. Theuse of protoplasts is, however, hampered by the regeneration systemwhich is highly species dependent. For transformation, protoplasts canbe used in conjunction with PEG to alter the plasma membrane whichcauses reversible permeabilization that enables the DNA to enter thecytoplasm as was demonstrated, for example, in Lolium multiform(Potrykus et al., 1985) and Triticum monococcum (Lörz et al., 1985).Another technique to increase the permeability of plasma membranes andeven cell walls to DNA is by electroporation (for review see Jones etal., 1987). In this method electrical pulses enable the DNA to enter thecells. Rice was the first crop in which fertile transgenic plantsresulted from protoplast electroporation (Shimamoto et al., 1989).

The use of particle bombardment or biolistics to deliver foreign DNAprovides an alternative method in cassava transformation. Particlebombardment is the only procedure capable of delivering DNA into cellsalmost in any tissue. The first transgenic plant obtained by using thismethod was in tobacco (Klein et al., 1989). Following this successfultransformation method, particle bombardment is widely used in plantswhich are less amenable to Agrobacterium infection, particularlymonocots. Improvement of several DNA delivery devices to accelerate theparticle (microprojectile) has resulted in the most recent model theBiolistic™ PDS-1000 (Bio-Rad Laboratories, Richmond, Calif.). Thosedevices are available commercially, however the price is relatively highat present. Tungsten or gold particles, coated with DNA, are commonlyused as microprojectiles to deliver DNA into the target tissue (recentlyreviewed by Songstad et al., 1995).

Selection and Reporter Genes Used in Genetic Modifications

To be able to identify transformed cells, the gene of interest iscoupled to a selectable marker gene. This marker gene is necessary toselect transformed cells. Selection can be based on a visualcharacteristic of the transformed cell/tissue. An example is theluciferase gene isolated from the firefly. Plant cells expressing thisgene and supplied with substrate (luciferin) will emit light which canbe detected with special equipment (Ow et al., 1986). Another way toselect transformed tissue is the introduction of a gene which encodesresistance to antibiotics or herbicides (Thompson et al., 1987;Gordon-Kamm et al., 1990).

A number of antibiotics and herbicides has been used as selective agentin plant transformation. In cereals resistance to the herbicidephosphinothricin (PPT) was chosen for the selection of transgenic plants(Cao et al., 1990). In Carica papaya (Fitch et al., 1994), Vitisvinifera (Nakano et al., 1994; Scorza et al., 1995), maize (Rhodes etal., 1988) and rice (Chen et al., 1987) the neomycine phosphothansferase(NPTII) gene, which confers resistance to kanamycin and relatedantibiotics (Fraley et al., 1986), was used as a selectable marker.

In cassava all above-mentioned systems of selection can be used, howeverPPT based selection has as advantage that it improves the ability of FECto form mature embryos and in this way increase plant regeneration.

TABLE 1 Genotypes of cassava used for somatic embryogenesis. IndonesiaNigeria TMS90853 M. Co122, Adira 1 TMS50395 TMS30555 Zimbabwe TjurugTMS60444 TMS30211 Line 11 Adira 4 TMS90059 TMS30395 Venuzuela Mangi 4TMS30572 TMS30001 M.Ven77 Gading TMS4(2)1244 Columbia Brasil FarokaTMS60506 M. Col 1505 Sao Paolo

TABLE 2 Influence of light intensity during growth of donor plants invitro on the number of leaf explants responding with the formation ofmature embryos and the number of mature embryos per cultured leafexplant (#ME/CLE). light intensity number of responding production(μEm⁻²s⁻¹) explants explants^(a) (# ME/CLE^(b)) 40 48 18 b 1.7 b 28 48 26 ab  4.9 ab 8 48 31 a 6.6 a ^(a,b)means with the same letter are notsignificantly different by respectively Chi-square test (p < 0.1) and byLSD test (p < 0.1)

TABLE 3 Influence of 2,4-D pretreatment on production of primary matureembryos (# mature embryos per cultured leaf explant isolated from invitro plants), followed by the multiplication of mature embryos bysecondary somatic embryogenesis in 11 Nigerian cassava genotypes and inM.Co122. embryogenesis primary^(a)) 2,4-D pretreatment no yessecondary^(b)) M.Co122 3.5 9.4 13.5 TMS 30555 0 0.7 6.2 TMS 50395 0 <0.15.3 TMS 60506 0 <0.1 0 TMS 90059 0 <0.1 7.2 TMS 30211 0 0 — TMS 60444 01.1 9.9 TMS 30395 0 0.1 6.7 TMS 90853 <0.1 0.2 8.2 TMS 4(2)1244 <0.1 05.4 TMS 30001 0 0 — TMS 30572 0 0 — ^(a))average of three experiments(total 48-74 leaf explants), ^(b))average of two experiments (total24-48 ME explants).

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What is claimed is:
 1. A method for producing protoplasts of cassavacomprising producing friable embryogenic callus from explants of cassavaand isolating protoplasts from said friable embryogenic callus, whereinthe protoplasts are capable of regenerating into complete plants.
 2. Amethod according to claim 1 whereby the friable embryogenic callus issubjected to culture in a liquid medium.
 3. A method according to claim1 whereby a mixture of cell wall degrading enzymes are used to produceprotoplasts.
 4. A method according to claim 2 whereby a mixture of cellwall degrading enzymes are used to produce protoplasts.
 5. A methodaccording to one of claims 1-4 whereby the plants from which theexplants are taken are pretreated with an auxin.
 6. A method accordingto one of claims 1-4 whereby the friable embryogenic callus is producedfrom torpedo shaped primary or mature embryos.
 7. A method according toclaim 6 whereby the embryos are induced on primary explants.
 8. A methodaccording to claim 3 wherein the enzymes are a cellulase, a pectolyaseand/or a macerozyme.
 9. A method for producing protoplasts of cassavacomprising: (a) inducing embryogenesis on cassava explants to produceembryogenic tissue; (b) producing friable embryogenic callus from saidembryogenic tissue; and (c) isolating protoplasts from said friableembryogenic callus, wherein the protoplasts are capable of regeneratinginto complete plants.